Among the first uses of gene therapy for monogenic disorders that demonstrated unequivocal substantial long term clinical benefit were for two forms of severe combined immune deficiency, SCID-X1 (caused by mutations in the IL2RG gene) and ADA-SCID (caused by mutations in the gene encoding adenosine deaminase). The approaches taken to achieve successful gene therapy for SCID-X1 and ADA-SCID, and the problems encountered have had a substantial impact on informing the past and ongoing initiatives to develop clinically beneficial gene therapy for CGD.
The first clinical trial of gene therapy for CGD occurred in 1995 and was directed toward young adult CGD patients with the p47phox deficient autosomal recessive form of CGD [25
]. The MFGS gene transfer vector (derived from murine Moloney leukemia virus) encoding p47phox cDNA was used to transduce peripheral blood mobilized autologous CD34+ cytokine-mobilized peripheral blood stem cells (PBSC) in a 4 day culture. Five patients were treated with a single cycle of gene therapy without any chemotherapy conditioning. Most received >0.5 × 106
transduced autologous CD34+ cells/kg. Based on the dihydrorhodamine flow cytometry (DHR) assay to measure reactive oxygen species (ROS) in individual neutrophils [26
], all five patients had the appearance of <1:2000 oxidase normal neutrophils in the peripheral blood. Trace numbers of oxidase normal neutrophils persisted for only a few months, and no clinical benefit was demonstrated. This trial pioneered the first clinical use of a closed system of gas permeable flexible plastic bags for culture and transduction of CD34+ hematopoietic stem cells (HSC) [27
In 1998 a clinical trial of similar design was initiated at the National Institutes of Health (NIH), also without conditioning, using the same MFGS retrovirus vector backbone encoding gp91phox and autologous CD34+ PBSC to treat 5 older teenage or young adult patients with X-linked CGD (X-CGD)[28
]. The much higher titer of vector and the application of Retronectin® (fibronectin fragment) coating of the inner surface of the culture bags resulted in ex vivo transduction efficiencies of >60%. Furthermore, patients received 1-4 cycles of treatment of >10 × 106
transduced autologous CD34+ cells/kg/cycle. Most patients had transient appearance of up to 1:900 oxidase normal neutrophils, which persisted in the circulation for only a few months with equivocal clinical benefit. Despite extraordinarily high bulk transduction rates and the large number of transduced autologous CD34+ PBSC infused, gene marking never exceeded 0.2%. However, the individual gene marked neutrophils in the circulation appeared to produce normal levels of ROS. Thus, the critical problem appeared to be that in the absence of bone marrow conditioning, circulating gene-corrected neutrophils were very low and transient.
Concordant with this second CGD clinical trial, investigators in France had begun to treat infants with SCID-X1 using a very similar MFG vector backbone encoding the IL2RG gene cDNA to transduce bone marrow CD34+ HSC [29
]. Despite the facts that the culture and transduction conditions for the SCID-X1 trial were similar to those in the two CGD trials, that rates of bulk ex vivo transduction averaged only 40%, that the number of transduced HSC infused were less than in the second CGD trial, that the SCID-X1 infants received only a single cycle of treatment, and that no conditioning was given, the clinical results were spectacularly better. The great majority of treated SCID-X1 infants developed normal numbers of functionally corrected T lymphocytes. There was some production of functionally normal B lymphocytes and even detection of some NK cells for a period of time. Investigators in London confirmed these results in a second trial [30
]. The general consensus for why there was such a successful outcome with SCID-X1 is that the substantially “empty” T lymphocyte compartment in SCID-X1 provides a setting where gene corrected T lymphocytes, with no competition from host T lymphocytes, have no barrier to peripheral growth and can expand to fill this empty hematologic niche. For B lymphocytes the hematologic niche occupied by this cell type is not “empty”; as it is occupied by B cells that cannot mature to produce antibody, suggesting why the correction of B cell immunity in SCID-X1 is substantially less than for T cell immunity. Even this cannot be the full explanation because these investigators found a persistent modest level of gene marking in the myeloid compartment of the infants where there should be no selective growth advantage. One can speculate that infants likely have a high marrow turnover rate that additionally facilitates substantially more efficient engraftment of gene marked autologous HSC than older children or adults.
Of the 20 patients with SCID-X1 achieving substantial immune reconstitution in the Paris and London gene therapy studies, 5 have had vector insertional mutagenesis-associated clonal lymphocytic leukemia [31
]. This has raised concerns about the safety of the genome insertion pattern of murine retrovirus vectors and in particular about the potential activation of nearby proto-oncogenes from this type of vector's long terminal repeat (LTR).
One of the earliest series of clinical trials of ex vivo gene therapy beginning in the early 1990's was the attempted treatment of children with ADA-SCID [33
]. While there was prolonged gene marking, possibly some improvement of immune function, and possible clinical benefit, none of the patients achieved a profound outgrowth of gene corrected T lymphocytes, substantial improvements in immune function, or significant clinical benefit. In 2002 investigators from Italy reported the results of a clinical trial of gene therapy for ADA-SCID that did achieve significant immune reconstitution and clinical benefit by using non-myeloablative conditioning with low dose busulfan (4 mg/kg total) prior to infusion of gene corrected autologous CD34+ HSC [35
]. This was a critical conceptual breakthrough demonstrating that in settings where selective growth advantage is less evident, marrow conditioning to enhance engraftment of gene corrected HSC can substantially improve the clinical outcome. These investigators also withheld enzyme replacement therapy with PEG-conjugated ADA in order to further enhance the growth advantage of gene corrected cells [36
]. These results were confirmed by studies in London and the U.S. [37
]. To date, no vector insertion mutagenesis related adverse events have been seen in the nearly 20 ADA-SCID patients treated with murine retrovirus gene therapy.
Gene correction for CGD should not provide any growth advantage either within the stem cell or myeloid compartments affected by the immune defect. Borrowing the lesson about conditioning from the ADA-SCID experience, investigators in Germany initiated a clinical trial of ex vivo gene therapy for X-CGD that incorporated non-myeloablative conditioning with a higher dose of busulfan (8 mg/kg total) to treat two adults [38
]. They used retrovirus vector derived from murine spleen focus forming virus (SFFV vector) encoding the human gp91phox cDNA to transduced autologous CD34+ PBSC, achieving about 40% transduction efficiency. Initially, there was 20-30% gene marking with significant levels of ROS activity in circulating neutrophils. Unexpectedly, the level of gene corrected neutrophils increased over six months until gene marked neutrophils comprised more than half of the circulating neutrophils. However, this temporal increase in gene marking was associated with oligoclonal expansion. The predominant clone in both patients had the vector inserted into MDS1/EVI1, where activation of this gene was likely responsible for the expansion. Initially, this benefited these patients in that their infections cleared. However, over the next 2 1/2 years, both patients developed myelodysplasia with monosomy 7 and loss of oxidase function in the gene marked myeloid cells (silencing), though with persistence of activity of the SFFV enhancer sequence [39
]. One patient died of severe sepsis likely related to the myelodysplasia, while the other was successfully treated with an allogeneic HSC transplant. Subsequently, a child with X-CGD achieved cure of a severe infection following gene therapy with the same SFFV vector, but without oligoclonal or clonal expansion [4
]. Nonetheless, concern for the safety of the SFFV vector has been raised.
In 2006 the NIH group used the same MFGS-gp91phox vector to treat X-CGD as in their 1998-2001 study, but used conditioning with non-myeloablative busulfan (10 mg/kg total) before infusing the transduced autologous CD34+ HSC [40
]. Interestingly, one patient in this trial previously had been treated in the 1999 trial with the same vector but without conditioning, affording an opportunity to evaluate the efficacy of busulfan conditioning. In 1999 this patient achieved gene marking of ~1:1500 (~0.75%) circulating neutrophils and the marking became undetectable after a few months. In 2006 this patient achieved gene marking of 24% of all circulating neutrophils at 3 weeks post infusion. This decreased to ~10% by three months and to ~1% at eight months after gene therapy. Strikingly, about 0.8% of circulating neutrophils in this patient are marked at almost 4 years after gene therapy (). Of particular note is that the individual gene marked neutrophils still demonstrate normal production of ROS without any evidence for silencing. An important conclusion from this study and the CGD study from Germany is that busulfan 8-10 mg/kg is likely adequate to achieve the necessary level of engraftment of gene-transduced HSC to correct a disorder such as CGD where there is no selective growth advantage in gene-corrected cells. The critical need is to find a vector with a high safety profile and improved transduction efficiency of HSC.
Figure 2 Dihydrorhodamine flow cytometry dot plot analysis of oxidase activity in phorbol ester stimulated circulating blood neutrophils of a patient in the NIH study with X-CGD who had busulfan conditioning followed by infusion of autologous CD34+ HSC transduced (more ...)
Most of the current gene therapy research focus for CGD is on lentivirus vectors because they may have a safer pattern of preferred insertion sites than murine retrovirus vectors. More important, lentivectors are more easily designed to incorporate 3′ LTR deletions resulting in self-inactivation of the LTR at the 5′ end following insertion into the genome of the target cell [41
]. This self-inactivating structure of the current generation of lentivectors may be intrinsically safer with respect to insertional mutagenesis mediated adverse events than the standard murine retrovirus vectors. However, more clinical experience with self-inactivating lentivirus vectors is necessary.
The recent report of clinically beneficial functional correction of X-linked adrenoleukodystrophy with high level persistent gene marking of myeloid cells using a lentivirus vector provides compelling evidence that they may result in intrinsically higher levels of gene transfer into HSC [42
]. Furthermore, in the adrenoleukodystrophy study, myeloablative conditioning was used and some have concluded that for future gene therapy trials for disorders with no intrinsic selective growth advantage conferred by gene correction, e.g. CGD, that not only should lentivectors be used, but that myeloablative conditioning is necessary. The counter-argument is that CGD patients who are candidates for gene therapy likely have ongoing infections, making use of ablative conditioning an unacceptable risk; and that previous trials of gene therapy for CGD suggest that sub-ablative conditioning may be sufficient. In addition, the use of myeloablative chemotherapy and resulting increase in early and late side effects reduces at least some of the attraction of the gene theapy approach compared to allogeneic HCT.
Self-inactivating lentivectors require the construct to have an internal promoter to drive production of the therapeutic transgene. The strongest internal promoters are virus promoters, but for safety reasons an adequately functioning mammalian gene promoter is preferable for CGD. This is problematic in designing lentivectors for X-CGD because most mammalian internal promoters to date are not sufficiently active to drive adequate production of gp91phox from the transgene to achieve functional correction of gene marked X-CGD neutrophils. Codon optimization of the gp91phox cDNA seems to help [43
], but this alone is insufficient. Current effort in lentivector development for X-CGD is focused on finding the best internal promoter to achieve functional correction of the gene marked neutrophil. An example of a candidate lentivector under development to treat X-CGD is shown in .
Figure 3 Candidate clinical insulated self-inactivating lentivirus vector plasmid pCL20i4r-EF1a-gp91OPT under development for treatment of X-CGD (unpublished data: HL Malech, EM Kang, U Choi, SS De Ravin, BP Sorrentino, RE Throm, and JT Gray). Shown is the vector (more ...)